Pasapera A M, Gutiérrez-Sagal R, García-Becerra R, Ulloa-Aguirre A, Savouret J F
Research Unit in Reproductive Medicine, Hospital de Gineco Obstetricia Luis Castelazo Ayala, Instituto Mexicano del Seguro Social, México DF.
Endocrine. 2001 Dec;16(3):217-25. doi: 10.1385/ENDO:16:3:217.
New and more potent progestins and antiprogestins suitable for reproductive therapy and contraception are currently the target of intensive research. The design of such drugs has been hampered by the complex technology required for screening these compounds at the molecular level. To solve this problem, we developed an in vitro cell system that allows detection of the progestagenic effects of a given compound using a PRE2-TATA-CAT reporter vector transiently introduced in a cell line stably transfected with the rabbit progesterone receptor (PR). The African Green Monkey Kidney CV1 (AGMK-CV1) cell line was chosen because these cells do not express endogenous steroid receptors; the selected clone stably expressing the rabbit PR has been maintained in our laboratory for more than 2 yr without detectable losses in PR content and progestagenic response. The presence and function of the PR were assessed by immunohistochemical and saturation analyses as well as by monitoring transactivation of the PRE2-TATA-CAT reporter gene. In this cell line, the PR is expressed at a concentration of 0.170 fmol/mg of protein, and the receptor is localized within the cell nucleus in either the presence or absence of the potent synthetic progestin R5020. This PR-expressing cell system allowed study of the in vitro progestational activity of several 19-nor progestins. The antiprogestin RU486 inhibited CAT activity induced by R5020; norethisterone (NET), levonorgestrel (LNG), and gestodene (GSD) induced PRE2-TATA-CAT activity at concentrations similar to those of R5020, whereas NET A-ring-reduced metabolites induced CAT activity at an extent lower than (5alpha-NET) or similar (3beta,5alpha-NET) to that of the precursor compound. The PRE2-TATA-CAT induction by 17beta-estradiol was also analyzed and no crossreactivity was detected. However, when the ERE-VitA2-TK-CAT (estrogen-responsive element-vitellogenin A2-thymidine kinase promoter-CAT) reporter vector and the estradiol receptor alpha or beta were cotransfected, CAT activity was induced in the presence of 17beta-estradiol, and NET tetrahydro-reduced derivatives. The results indicate that this AGMK-CV1-PR cell assay system appears to be suitable for measuring the effects of different synthetic progestins at the transcriptional level. In this assay system, NET, LNG, and GSD exhibit potent progestational effects at the transcriptional level. In the particular case of NET, the assay system allowed us to determine that the single or multiple hormonal transcriptional effects of this compound are partially mediated by its A-ring-reduced derivatives.
新型且效力更强的适用于生殖治疗和避孕的孕激素及抗孕激素目前是深入研究的目标。这类药物的设计因在分子水平筛选这些化合物所需的复杂技术而受阻。为解决这一问题，我们开发了一种体外细胞系统，该系统可使用瞬时导入稳定转染兔孕激素受体（PR）的细胞系中的PRE2-TATA-CAT报告载体来检测给定化合物的孕激素作用。选用非洲绿猴肾CV1（AGMK-CV1）细胞系是因为这些细胞不表达内源性类固醇受体；所选稳定表达兔PR的克隆已在我们实验室保存了2年多，PR含量和孕激素反应未检测到损失。通过免疫组织化学和饱和分析以及监测PRE2-TATA-CAT报告基因的反式激活来评估PR的存在和功能。在该细胞系中，PR以0.170 fmol/mg蛋白质的浓度表达，且无论有无强效合成孕激素R5020，该受体均定位于细胞核内。这个表达PR的细胞系统可用于研究几种19-去甲孕激素的体外孕激素活性。抗孕激素RU486抑制R5020诱导的CAT活性；炔诺酮（NET）、左炔诺孕酮（LNG）和孕二烯酮（GSD）在与R5020相似的浓度下诱导PRE2-TATA-CAT活性，而NET A环还原代谢产物诱导CAT活性的程度低于（5α-NET）或与前体化合物相似（3β,5α-NET）。还分析了17β-雌二醇对PRE2-TATA-CAT的诱导作用，未检测到交叉反应。然而，当共转染ERE-VitA2-TK-CAT（雌激素反应元件-卵黄生成素A2-胸苷激酶启动子-CAT）报告载体和雌二醇受体α或β时，在17β-雌二醇和NET四氢还原衍生物存在的情况下诱导了CAT活性。结果表明，这种AGMK-CV1-PR细胞检测系统似乎适用于在转录水平测量不同合成孕激素的作用。在该检测系统中，NET、LNG和GSD在转录水平表现出强效孕激素作用。在NET的特殊情况下，该检测系统使我们能够确定该化合物的单一或多种激素转录作用部分由其A环还原衍生物介导。
